Work on this project has focused on three different types of skeletal dysplasia: (1) the achondroplasia family of skeletal dysplasias, (2) an autosomal recessive form of skeletal dysplasia, Cartilage Hair Hypoplasia (CHH), and (3) the Schwarz-Jampel syndrome. Our collaborative work with Dr. Yoshi Yamada and his colleagues at the NINDR has demonstrated that the Schwarz-Jampel syndrome is caused by mutations in the perlecan gene. The achondroplasia family of skeletal dysplasias includes three previously recognized diagnoses and one that has been defined under this project. The three well-established conditions are achondroplasia, hypochondroplasia and thanatophoric dysplasia (TD). Work published by our lab in the past described a new syndrome, Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN). All four disorders in this family of conditions are caused by mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3). Using a knock-in strategy, mice heterozygous for K644E and K644M mutations have been generated. Ongoing work is focused on creation of mice with K644N mutations, to model the human disorder hypochondroplasia. Over the past year, tissue-specific expression of the K644E mutation has been accomplished using mice carrying the cre recombinase under the control of tissue-specific promoters. These mice are assisting in assessing the contribution of mutant FGFR3 expression in the developing central nervous system to morbidity and mortality in these disorders. A manuscript describing these observations was published in Human Molecular Genetics. We anticipate using microarray studies to dissect the downstream pathways associated with pathogenesis of these three disorders over the coming year. Cartilage hair hypoplasia (CHH) has recently been found to be caused by mutations in the RNMP gene, which encodes an RNA that participates in the Mitochondrial Ribonuclear Protein complex. In collaboration with Dr. Douglas Wallace, we plan to explore the effects of the mutation on mitochondrial function during the coming year. In addition, over the coming year Dr. Shepherd Schurman and Joseph Tran will be working to create a mouse model for Cartilage Hair Hypoplasia In addition to our work on specific skeletal dysplasias, Dr. ZJ Zhang has been using in vitro methods to create and enhance an organ culture method for the development of cartilage. It is hoped that these studies will lead to the development of treatment strategies for mendelian and complex disorders of cartilage growth and development. Dr. Zhang has shown that pulsed low intensity ultrasound produces a demonstrable anabolic effect on matrix production and chondrocyte hypertrophy. Dr. Zhang's work describing the effects of pulsed low intensity ultrasound was published in 2003 in Ultrasound Mol Biol. Dr. Zhang has also been pursing studies designed to investigate the utility of pellet-culture for cartilage engineering, isolated chondrocytes were grown in pellet culture for 2 weeks. The chondrocyte phenotype was stabilized in this specially designed culture system as show by aggrecan staining and immunohistochemistry for type II collagen. During the culture period, chondrocytes maintained expression of type IX collagen, which functions to link collagen fiber with aggrecan. The reconstruction of extracellular matrix was demonstrated by electron microscopy. By 2 weeks, the fiber network in the engineered cartilage was similar to the native cartilage at fiber diameter and density, but the fiber orientation was less organized. A manuscript describing this effort is in press at the time of this report (September 2004). The HGIMS is also continuing to maintain the Skeletal Gene Database, a publicly available electronic resource cataloguing all genes and expressed sequences found in skeletal tissue, and all known associations between genes and disease states or traits affecting the skeletal system. Over the past year, a clinical protocol to examine cardiovascular and pulmonary risk factors in adults with achondroplasia has been approved and implemented. Over the next 3 years, it is anticipated that 150 subjects with achondroplasia will be enrolled in this study.